Traumatic resin-canal formation caused by inoculation withCistella japonica in secondary phloem ofChamaecyparis obtusa

In the resinous lesion development caused by the inoculation withCistella japonica onChamaecyparis obtusa stem, numerous traumatic resin-canals were formed at certain radial intervals in the secondary phloem. These formation started in the ring grown in the current or the previous year of inoculation and the resin-canals were subsequently formed in the newly grown rings. The resin-canals were arranged in tangential series in one to two lines in an annual-ring. Artificially induced resin-canal formation was similar to that in naturally infected resinous stem canker. The results from the present study reconfirmed thatCi. japonica is the causal agent of the disease.     

Etiology of the resinous stem canker ofChamaecyparis obtusa: Cistella japonica as the causal agent

In order to confirm the pathogenicity ofCistella japonica, inoculation experiments were conducted onto several coniferous trees. Resinous lesions similar to those of the resinous stem canker developed by the inoculation withCi. japonica only onChamaecyparis obtusa. Chamaecyparis trees were heavily affected when inoculated with the fungus in November, but slightly in May and August. Resin flows occurred when inocula ofCi. japonica were put into the holes made on stem barks and on pruning scars. Isolates ofCi. japonica varied greatly in the pathogenicity. On the lesions caused by the fungus, resinous and necrotic areas expanded in fusiform or oblong shape, and resin cysts were formed in inner barks. Beneath some necrotic inner barks, sapwood was suppressed in growth for the damages in cambial cells and stained.Ci. japonica was reisolated from the inoculated lesion tissues, though isolation frequency of the fungus became lower in some lesions two or more years after the inoculation. The results of these inoculations proved thatCi. japonica was the causal agent of the resinous stem canker ofCh. obtusa.     

Progeny differences of hinoki (Chamaecyparis obtusa) and sawara (C. pisifera) against resinous stem canker disease and spatial distribution of damage (disease severity) in a progeny test

The incidence and severity of resinous stem canker disease were investigated in hinoki (Chamaecyparis obtusa) and sawara (C. pisifera) at a progeny test located in Yamatsuri Town, Fukushima Prefecture, Japan. Symptoms of the disease were observed in 307 trees out of 933 investigated trees (32.9%). The damage was more severe on lower slopes than on upper slopes, indicating that micro-environmental factors are causally associated with the occurrence of the disease. The severity of the disease varied both among nine open-pollinated progenies derived from hinoki plus-tree clones and among 13 progenies derived from pollination between hinoki plus-tree clones and the mixed-pollen of hinoki plus-tree clones. The severity of the disease also varied with height above the ground. This tendency was most obvious where the disease was most severe. An estimation of the narrow sense heritability,h ², of 0.214, was obtained from analysis of the 13 progenies derived from pollination with mixed-pollen. The heritability derived from material that had not been subjected to selection against the disease, indicated that breeding to enhance resistance would be possible to control the disease. The interaction between environments and progenies in relation to severity of the disease is also discussed. Ms. Setsuko Chiba and Mr. Mamoru Ueta gave us vigorous supports, and Mr. Haruki Orita, Dr. Tomiyasu Miyaura, and Dr. Shinichiro Ito gave us many helpful comments. We deeply appreciated their cooperation. The study was supported by a grant from the Ministry of Agriculture, Forestry. and Fisheries of Japan.     

A new diterpene dimer from the bark ofChamaecyparis obtusa

This paper describes characterization of diterpenes of the bark of Japanese cypress,Chamaecyparis obtusa (S. and Z.) Endl, without the resinous stem canker to learn the difference between the cypress bark affected with the canker. A diterpene dimer and two diterpenes, 6,12-dihydroxyabieta-8,11,13-trien-7-one and 6,12-dihydroxyabieta-5,8,11, 13-tetraen-7-one, were firstly isolated from Japanese cypress. The dimer, 6-(abieta-6,8,11, 13-tetraenyl-12-oxy)-7-methoxyabieta-8,11,13-trien-12-ol, was a new compound. It is a terpene indicative ofC. obtusa not infected with the resinous stem canker. Five known diterpenes were also isolated.     

Comparison of terpenes in extracts from the resin and the bark of the resinous stem canker of Chamaecyparis obtusa and Thujopsis dolabrata var. hondae

A monoterpene and 15 diterpenes were isolated from the ethyl acetate extracts of the bark-glued resin from the resinous stem canker ofThujopsis dolabrata var.hondae Makino. A monoterpene (nezukone20) and 4 diterpenes (acetyl torulosol5, acetyl isocupressic acid8, acetyl abietinol11, and 7-methoxytotarol18) were characteristic constituents of the ethyl acetate extracts but were absent in then-hexane extracts from the resinous stem canker ofT. dolabrata var.hondae. These terpenes were first isolated fromT. dolabrata var.hondae andT. dolabrata. The available literature suggests that diterpene18 is a new compound. The resinous stem canker ofChamaecyparis obtusa Endlicher contained larger amounts of manool1,trans-communic acid6, and ferruginol12 and smaller amounts of isocupressic acid7 and abietinol10 than the resinous stem canker ofT. dolabrata var.hondae. The concentration of18 was less than 2% in the extracts, and the resinous stem canker ofC. obtusa lacked this compound. The resinous stem cankers ofC. obtusa andT. dolabrata var.hondae provided extracts 15.6 and 4.96 times, respectively, heavier than the healthy ones. Large differences in the ratios and compositions of terpenes were also observed between the resinous stem canker and the healthy trees. Terpenes isolated from the extracts contained many kinds of diterpene, especially the labdane-type diterpenes, in these diseased trees. These results suggest that the presence of labdane-type diterpenes is closely associated with the resinous stem canker or the causal fungi of this disease.Part of this report was presented at the 50th annual meeting of the Japan Wood Research Society, Kyoto, April 2000     

Methods for inoculum production and inoculation of Cistella japonica,the causal agent of resinous stem canker in Chamaecyparis obtusa

The ascomycete Cistella japonica was cultured on potato dextrose agar medium (PDA) for inoculation into Chamaecyparis obtusa, enabling the development of an inoculation method suitable for use in a breeding programme aimed at selecting for families of Ch. obtusa resistant to resinous stem canker. Using PDA to generate the inoculum resolved the problems encountered with the previously used mixed medium of rice bran and wheat bran, including unfavourable characteristics, uncertain growth of Ci. japonica mycelia, and a complex culturing operation. The inoculation test induced resin exudation similar to that observed in natural infections, and reproduced clonal differences with regard to damage severity.     

Individual variation in low boiling point monoterpenes emitted from hinoki (Chamaecyparis obtusa) needles

Variations in the compositions of low-boilingpoint (LBP) monoterpenes in needle samples of 50 hinoki (Chamaecyparis obtusa) trees were investigated using the headspace technique. Considerable compositional variations were revealed, especially in sabinene composition. The sabinene composition varied from 4.9% to 78.0% of the total LBP monoterpenes.-Pinene, myrcene, and limonene also showed considerable variations (9.0%–32.7%, 5.5%–22.6%, 3.6%–29.0% respectively). Analysis of the monoterpene composition allowed definition of four chemotypes based on the contingency table test. No correlation was observed between tree size and LBP monoterpene composition, indicating that the compositional variation in LBP monoterpene exists genetically in this population of hinoki.Part of this paper was presented at the 11th annual meeting of the Chugoku Shikoku branch of the Japan Wood Research Society, Matsue, September 1999     

New lignan, isoactifolin, fromChamaecyparis obtusa cv. Breviramea

A new lignan isoactifolin was isolated from young shoots (with leaves) ofChamaecyparis obtusa cv. Breviramea. The structure of the compound was determined based on spectroscopic evidence.Parts of this report were presented at the 44th lignin symposium, Gifu, October 1999     

Water status of hinoki cypress, Chamaecyparis obtusa, attacked by secondary woodboring insects after typhoon strike

We estimated the water status of six 31-year-old hinoki cypress (Chamaecyparis obtusa) trees that were struck by a large typhoon. Measurements were made of the trunk water-conduction area of xylem, leaf water potentials, and diurnal changes in trunk diameters. The secondary woodboring insects, Callidiellum rufipenne and Ovalisia vivata, on the trees were captured by the sticky-trap banding method. In trees on which these wood borers were captured, water conduction areas were reduced, leaf water potentials were lower, and trunk diameter changes (R) fluctuated during the day, suggesting that the trees had been damaged by the typhoon and were under water stress. The secondary woodboring insects attacked the water-stressed trees.     

Chemical composition and antimicrobial activity of Chamaecyparis obtusa leaf essential oil

The essential oil obtained from the leaves of Chamaecyparis obtusa was analyzed by GC and GC-MS. alpha-Terpinyl acetate, sabinene, isobornyl acetate and limonene were found to be the major components. The oil showed relatively strong antibacterial activities against Gram (+) bacteria and some fungi.     

Liquid culture and transformation of hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.)

Hinoki cypress (Chamaecyparis obtusa) is one of the most important timber resource forest trees in Japan. Because seed production from a seed orchard of hinoki cypress is not constant every year, micropropagation from a limited amount of material is useful. Up to now, the conventional tissue culture method using solid medium has been used. Here a new method using liquid culture in tubes rotated vertically is described. Shoot primordium of hinoki cypress was inoculated in Campbell and Durzan’s (CD) liquid medium containing different cytokinins (6-benzylaminopurine (BAP), Zeatin, thidiazurone (TDZ)), and the container tubes were rotated vertically around the axis at 2 times / min. Culture room temperature was 25°C and light condition was 16 h photoperiod per day of fluorescent lamps. Zeatin at 1μM concentration was the best for maintaining the shoot primordium production and TDZ induced callus on the surface of the shoot primordia. After shoot primordium multiplication in the liquid culture, they were transplanted to agar medium for shoot elongation. A high concentration of agar (up to 16 g/L) or AVF (anti vitrification factor from Dr. Nairn, 1995) was effective to prevent vitrification of the shoots. Transformation of shoot primordium was done using particle bombardment with vectors containingβ-glucuronidase (GUS) gene or herbicide resistance gene (bar). Positive result for transient transformation was observed with the histo-chemical study for transformation with GUS. Integration of a useful herbicidebar gene into the shoot primordium culture system was also tried and stably transformed plants were obtained. This is the first report of stable transformation of Japanese conifer using practically useful gene. The generous supply of AVF-B from Dr. B.J. Nairn, Tasman Forestry, NZ is also appreciated.     

The penetration rate of laser pulses transmitted from a small-footprint airborne LiDAR: a case study in closed canopy, middle-aged pure sugi (Cryptomeria japonica D. Don) and hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.) stands in Japan

Penetration rates of laser pulses transmitted from a small-footprint airborne light detection and ranging (LiDAR) system in closed-canopy, middle-aged (40–50 years old) hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.) and sugi (Cryptomeria japonica D. Don) plantations that had similar levels of canopy openness were studied. The number of transmitted pulses was 107 427 points/ha for the hinoki cypress stand and 122 883 points/ha for the sugi stand. The penetration rates of the first (P_f) and second pulses (P_s) that reached the ground were 0.1% and 1.0% in the hinoki cypress stand, and 2.6% and 5.5% in the sugi stand, respectively. Penetration rates for the total number of pulses (P_f+s) that reached the ground within each stand were 1.1% and 8.1%, respectively, and values for P_f+s, P_f, and P_s for sugi and hinoki cypress stands were significantly different (P < 0.001). The results of this study indicated that the generation of accurate digital terrain models in dense hinoki cypress stands with complex topographies using only the data from laser pulses with poor laser penetration rates is likely to be difficult.     

The effect of bark decortication for hiwada production on xylem and phloem formation in Chamaecyparis obtusa

Of all plant materials used to cover the roofs of traditional Japanese buildings, Japanese cypress (Chamaecyparis obtusa) bark, hiwada, has the longest service life and has been used from ancient times. However, wood and bark properties after hiwada harvest have not been evaluated in detail. We studied whether decortication for hiwada production in winter affected xylem and phloem formation. Decorticated trees still preserved all inner bark and part of the outer bark, and both decorticated and control trees had similar annual ring structures at all stem heights in the xylem and phloem. In both xylem and inner bark, no significant difference in ring width at any stem height was found between annual rings before and after decortication. Thus, this study revealed that the decortication of bark for hiwada production does not affect the formation of xylem and the inner and outer bark if decortication is carried out by highly skilled workers in winter.     

Detection of genetic variation among seed and seedlings ofChamaecyparis obtusa using allozyme markers

The effects of nursery practices on genetic composition and structure were studied inChamaecyparis obtusa by analyzing seeds and one-, two-, and three-year-old seedlings. Enzyme polymorphisms of the6Pgd-2, G6pd, Got, Gk, Shd-2, Pod, Dia-1, andPgm loci were used. OnlyDia-1 indicated significantly different allelic frequencies between seeds and three-year-old seedlings. Most of the genetic diversity of the parental clones in this seed orchard could be transferred to the offsprings. Genetic diversity on the basis of an average expected heterozygosity did not differ between seeds and seedlings. A slight excess of homozygotes in seeds and a significant excess of heterozygotes in seedlings of different ages to the expected panmictic proportions were found. Most of the differences in genotypic distributions among the seed and seedling stages, probably resulted from viability selection favoring outcrosses, and selective removal of inbred offsprings including self-fertilized ones prior to their transplantation in the nursery. A part of this paper was orally presented at the 107th Annual Meeting of the Japanese Forestry Society (1996).     

Butt rot ofChamaecyparis obtusa (Sieb. et Zucc.) Endlicher trees caused byPerenniporia subacida in Shikoku district, Japan: Pathogen, distribution of damaged trees in the stand, and soil investigation

The rot ofChamaecyparis obtusa (hinoki) trees was studied in the northern part of Kagawa Prefecture, Japan. Among 158 hinoki examined, butt rot was found in 28.6, 58.3, and 100% of trees in the 29, 30, and 34-year-old stands, respectively. All trees with butt rot, and 11 trees without it had rotted roots. Thirteen hinoki were peeled byCervus nippon (sika deer) and all of them were infected with butt rot. The maximum height of rot in deer-damaged trees was 2.6 m from the ground level, but was 1.9 m in undamaged trees. About eighty-two percent of rotted trees showed rotted areas of less than 40 cm² on the cross section of stems at the ground level. White mycelia and black flecks sometimes appeared in the rotted wood. Basidiocarps of polyporaceous fungus were often found on felled logs and rotted stumps of hinoki and identified asPerenniporia subacida. Basidiomycetous fungus was isolated frequently from rotted wood of roots and stems, and determined to beP. subacida by comparative study on cultural characteristics. An inoculation experiment and wood-decay test proved that the fungus was the cause of the rot of hinoki. Few absorbing roots of living trees were found in the clayey subsurface soil of the high dry bulk density and the less soil aeration. Some absorbing roots had root rot and the rot spread from the base of the absorbing root to the central part of the woody root. This is the first report on the rot of hinoki caused byP. subacida in Japan.     

Efficient plant regeneration of Hinoki cypress (Chamaecyparis obtusa) via somatic embryogenesis

This report describes the efficient plant regeneration of Chamaecyparis obtusa Sieb et Zucc. via somatic embryogenesis. Embryogenic cultures were initiated from megagametophytes containing immature zygotic embryos. Embryogenic cultures were maintained and proliferated by 2–3-week interval subcultures in medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. High maturation frequencies of cotyledonary embryos were obtained on maturation medium containing maltose, polyethylene glycol, activated charcoal, and abscisic acid. Somatic embryos germinated readily after transfer to plant growth regulator-free medium. Growth of regenerated emblings has been monitored in a greenhouse.     

Pollen dispersal in a hinoki (Chamaecyparis obtusa) seed orchard detected using a chloroplast DNA marker

Pollen dispersal was estimated in two test plots in a hinoki (Chamaecyparis obtusa) seed orchard using a chloroplast DNA marker, the spacer region between thetrnD andtrnY genes, and SSCP (single strand conformation polymorphism). In Plot 1, 2,020 seeds from 40 trees within 30 m of the marker tree were analyzed using the PCR-SSCP method. In Plot 2, 1,850 seeds from 37 trees were analyzed in the same manner. The results revealed that the maximum pollen dispersal distance in the two plots exceeded 25 m. Pollen dispersal appeared to be inversely proportional to the distance from the marker tree. The effective pollen dispersal was suggested to be less than about 20 m in a mature hinoki seed orchard. Adjacent trees had an excessive influence when the pollen density was increased by artificial flower stimulation. Therefore, it was suggested that seed production better resembles ideal random mating when carried out as naturally as possible. In conclusion, the SSCP chloroplast DNA marker was a useful tool for amassing basic information on pollen management in seed orchards of coniferous species.     

Seasonal change in the temperature coefficientQ 10 for respiration of field-grown hinoki cypress (Chamaecyparis obtusa) trees

The effect of temperature upon nighttime respiration was examined on four different sized sample trees in a 17-year-old hinoki cypress (Chamaecyparis obtusa (Sieb. et Zucc.) Endl.) stand over two years. Seasonal changes inQ ₁₀ values and their responses to mean temperature were investigated. On the basis of the monthly relationships between nighttime respiration (r) and temperature inside a chamber (θ),r=r ₀exp (kθ), theQ ₁₀ value (=exp(10k)) was calculated. TheQ ₁₀ values were high (Q ₁₀≥3.0) in winter when mean air temperature was low, and gradually decreased toward summer (Q ₁₀≤1.5) through spring with increasing temperature. TheQ ₁₀ values were negatively correlated with mean air temperature. The response ofQ ₁₀ values to mean air temperature was described by a single equation, regardless of tree size. This result, which might be characteristic of this species, shows that respiration ofC. obtusa trees is promoted by slight increases of air temperature in winter season. On the other hand, temperature sensitivity of total respiration reduced during growing season when ambient temperature was high. These chaning temperature sensitivity according to seasons may depend on the seasonal change of the ratio of growth respiration to total respiration. It is concluded that changes in temperature due to changing seasons not only change respiration rate, but also change the response of respiration rate to temperature by shiftingQ ₁₀ values.     

Genetic variation in fruitfulness in a hinoki (Chamaecyparis obtusa Endl.) seed orchard and its impact on the maintenance of genetic diversity in seedlots

Genetic variation in seed/cone production among clones was studied in a hinoki (Chamaecyparis obtusa Endl.) seed orchard containing 25 plus-trees by analyzing the number of cones, the yield of cones and seeds of individual ramets for 5 successive years (1982 to 1986). There was significant variation among clones each year and parental contribution in the seed orchard. Specifically, in the years 1982, 1983, 1984, 1985, and 1986, 20% of the clones produced 37.2, 60.6, 36.0, 44.3, and 44.8% of the total cones, respectively. The size of the crop greatly influenced the parental balance in the resulting seed/cone crops. The product moment correlation coefficients and Spearman’s coefficients of rank correlation were small and insignificant between consecutive years, but large and highly significant between alternate years, suggesting the presence of carry-over effects in seed/cone production. The broad-sense heritability on a clone mean basis was 0.74 ± 0.15 for the number of cones, 0.72 ± 0.14 for the yield of cones, and 0.68 ± 0.13 for the yield of seeds. The corresponding heritabilities from analyses combined over all years were 0.24, 0.558, and 0.724, respectively. These results indicate that seed/cone production in hinoki is under strong genetic control. Several managerial measures are discussed that maintain the genetic diversity in seedlots used for reforestation, by reducing the variation in seed/cone production among clones and producing seed crops with equal contributions from all parents.     

Distinction of resin compounds between the healthy bark and the resinous stem canker of <Emphasis Type="Italic">Thujopsis dolabrata</Emphasis> var. <Emphasis Type="Italic">hondae</Emphasis>

Methyl esters of higher fatty acids were detected from the healthy bark of Thujopsis dolabrata var. hondae but not from the bark of the resinous stem canker of T. dolabrata var. hondae. This difference enabled us to distinguish healthy trees from diseased ones. Fourteen diterpenes were also isolated from the n-hexane extracts of the bark-glued resin taken from the resinous stem canker of T. dolabrata var. hondae. Of these diterpenes, abietane diterpenes [abieta-7,13-diene (1), abietinol (2), dehydroabietinol (4)], pimarane diterpene [sandaracopimaric acid (8)], and labdane diterpenes [manool (10), torulosol (11), torulosal (12), cupressic acid (13)] were first isolated from T. dolabrata var. hondae.Part of this paper was presented at the 47th Annual Meeting of the Japan Wood Research Society, Tokyo, April 1999